ABSTRACT The broad long term objective is to improve methods of disease treatment in the liver. Alcoholic liver disease (ALD) results from excessive alcohol consumption and is the cause of considerable morbidity and mortality world-wide. In the United States, ALD is a leading cause of GI-related deaths, with about half of the 75,000 liver disease deaths each year being related to alcohol use. Although there are few therapeutic options, a new lead has emerged from our studies of exosomes that are produced by normal healthy hepatocytes. Our Preliminary Data show that these exosomes (i) attenuate expression of genes that regulate fibrogenesis or activation in cultured primary hepatic stellate cells (HSC; the principal fibrosis-producing cell type in the liver); (ii) suppress fibrogenic pathways and reverse fibrosis in experimental models of hepatic fibrosis in vivo; (iii) bind more strongly to hepatocytes in injured livers than hepatocytes in control livers; and (iv) attenuate ethanol-induced cytotoxicity in primary cultured hepatocytes. Our overall objective is to establish therapeutic uses of exosomes for treating liver disease. Our central hypothesis is that exosomes from hepatocytes are therapeutic in mouse models of ALD. The Specific Aims to test this hypothesis are: Specific Aim 1: Determine that hepatocyte exosomes attenuate ethanol-induced liver injury Exosomes will be (i) administered to mice that receive ethanol for either 25 days (Lieber deCarli diet) or for 10 days followed by acute ethanol gavage (binge model) to determine their therapeutic effect on steatosis, inflammation, cell damage, or macrophage involvement; or (ii) added to cultured hepatocytes or Kupffer cells (KC) to determine their reversal of the effects of, respectively, ethanol or lipopolysaccharide (LPS), on viability, apoptosis, activation of MAPK or caspases, and production of inflammatory cytokines, ROS, or damage- induced danger signals. Specific Aim 2: Identify therapeutic microRNAs in hepatocyte exosomes Using an unbiased analysis, miRs will be identified that are the most differentially expressed between exosomes from normal versus ethanol-injured hepatocytes and for which the higher level of expression occurs in exosomes from normal hepatocytes. Candidate miRs, validated by PT-PCR, will be tested for their modulation of damage-inducing pathways in hepatocytes after ethanol/TNF-? treatment or in KC after LPS treatment. The expected outcome will be to establish a new exosome-based therapy for treating alcohol-induced cell damage and altered immune function that are key features of ALD pathogenesis. The rationale underlying the proposal is that exosomes produced by normal non-injured hepatocytes contain a molecular cargo, including miRs, that reflects their overall healthy status. The positive impact of these studies is that they will provide new knowledge to improve the health of millions of people world-wide with ALD or other chronic liver diseases